Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits.

Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF-factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 +/- 25 vs. 49 +/- 12 pg mg-1 protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 +/- 1 s) compared with the normal rabbits (44 +/- 1 s, P < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF-FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg-1. FVIIai dose-dependently reduced thrombus mass (14.7 +/- 2.5 and 5.9 +/- 2.2 mg, respectively, vs. 21.6 +/- 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF-FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF-FVIIa inhibitors.

Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma.

BACKGROUND: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. METHODS: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. RESULTS: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. CONCLUSION: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

Molecular analysis of complex tissues is facilitated by laser capture microdissection: critical role of upstream tissue processing.

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.

Hypercholesterolemia causes mechanical weakening of rabbit atheroma : local collagen loss as a prerequisite of plaque rupture.

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.

Collagen synthesis in atherosclerosis: too much and not enough.

Fibrillar collagen is a critical component of atherosclerotic lesions. Uncontrolled collagen accumulation leads to arterial stenosis, while excessive collagen breakdown combined with inadequate synthesis weakens plaques thereby making them prone to rupture. This review discusses cellular sources of collagen synthesis in atherosclerosis, local and systemic factors modulating collagen gene expression, as well as temporal and spatial patterns of collagen production in human and experimental atherosclerotic lesions.

Animal model that mimics atherosclerotic plaque rupture.

Atherosclerotic plaque rupture is the main cause of coronary thrombosis and myocardial infarcts. Currently, there is no animal model of plaque disruption. We have developed a rabbit model in which an atherosclerotic plaque can be ruptured at will after an inflatable balloon becomes embedded into the plaque. Furthermore, the pressure needed to inflate the plaque-covered balloon may be an index of overall plaque mechanical strength. The thoracic aorta of hypercholesterolemic rabbits underwent mechanical removal of endothelial cells, and then a specially designed balloon catheter was introduced into the lumen of the thoracic aorta. As early as 1 month after catheter placement, atherosclerotic plaque formed around the indwelling balloon. The plaques were reminiscent of human atherosclerotic lesions, in terms of cellular composition, patterns of lipid accumulation, and growth characteristics. Intraplaque balloons were inflated both ex vivo and in vivo, leading to plaque fissuring. The ex vivo strategy is designed to measure the mechanical strength of the surrounding plaque, while the in vivo scenario permits an analysis of the plaque rupture consequences, eg, thrombosis. In addition, our model allows local delivery of various substances into the plaque. The model can be used to study the pathogenesis of plaque instability and to design plaque stabilization therapy.

Graft permeabilization facilitates gene therapy of transplant arteriosclerosis in a rabbit model.

BACKGROUND: Smooth muscle cell (SMC) replication plays a central role in the pathogenesis of transplant arteriosclerosis. One strategy to eliminate dividing cells is to express a herpesvirus thymidine kinase (tk) gene that phosphorylates the nucleoside analogue ganciclovir into a toxic form leading to cell killing. However, medial SMCs are resistant to gene transfer unless the artery undergoes deendothelialization. We hypothesized that manipulations that increase the "porosity" of the artery can make SMCs prone to gene transfer without denudation. METHODS AND RESULTS: In organ culture of rabbit aorta, longitudinal stretch and supraphysiological pressure applied for 3 hours during incubation with adenoviral vector facilitated gene transfer into medial SMCs without denudation. Of the SMCs, 10.2+/-3.8% expressed a reporter gene of human placental alkaline phosphatase (hpAP), whereas SMCs in control arteries did not express hpAP. To evaluate the feasibility of transgene expression in arterial grafts, we performed such permeabilization-assisted reporter gene transfer into aortas of donor Dutch Belted rabbits and transplanted them into carotid arteries of recipient New Zealand White rabbits. Unstretched transfected grafts were used as a control. SMCs expressed hpAP (7. 3+/-2.4% of cells in 2 days and 4.2+/-1.9% in 2 weeks) in stretched grafts only. In the next series of experiments, we transfected stretched grafts with ADV-tk and combined transplantation with systemic administration of ganciclovir. Stretched ADV-hpAP grafts were used as a control. In 2 weeks, the formation of intimal thickening in tk-expressing grafts was significantly reduced (P<0. 01) because of a decrease in proliferating SMCs. CONCLUSIONS: Manipulations within target tissues can enhance the efficiency of gene transfer into SMCs. Although mechanical permeabilization is clinically problematic, in principle, targeting SMC replication may provide a genetic approach to the treatment of transplant arteriosclerosis.

Gene transfer into normal and atherosclerotic human blood vessels.

Gene transfer to blood vessels is a promising new approach to the treatment of the vascular diseases, but the feasibility of gene transfer to adult human vessels has not been explored. We introduced an adenovirus vector encoding a marker gene human placental alkaline phosphatase into normal and atherosclerotic human vessels in organ culture. In the normal vessels, recombinant gene was expressed preferentially in the endothelial cells (approximately 100%), intimal smooth muscle cells (1.3+/-0.4%, 1.4+/-1.0%, and 3.8+/-0.8% in the internal mammary arteries, saphenous veins, and normal coronary arteries, respectively), and various adventitial cells. Advanced, complicated atherosclerotic plaques demonstrated a similar efficiency of recombinant gene expression (3.1+/-0.5% and 3.8+/-0.3% of nonendothelial intimal cells in the coronary artery and carotid artery plaques, respectively). Of these intimal cells, macrophages and smooth muscle cells expressed a transgene, identifying them as targets for gene transfer. Areas of plaque rupture and thrombus are sites of predilection for expression of recombinant genes. Collagenase and elastase treatment increased the percentage of transgenic alkaline phosphatase-positive cells 7 times (P<0.001), suggesting that the pattern of gene expression was affected by the amount of surrounding extracellular matrix. These studies demonstrate the feasibility of gene transfer to human blood vessels. However, these studies also highlight important barriers to adenoviral gene delivery to the actual normal and atherosclerotic human vessels of clinical interest.

Procollagen production in fresh and cryopreserved aortic valve grafts.

Long-term durability of aortic valve allografts may be enhanced by cellular capacities for regeneration and repair. To evaluate aortic valve graft production of an important structural protein, rat aortic roots were implanted heterotopically into the abdominal aorta of recipient rats. Grafts were either syngeneic or strongly allogeneic, were implanted either fresh or after cryopreservation, and were left in place 2 to 21 days after implantation. A total of 80 aortic valve grafts and the corresponding native aortic valves were examined. The grafts were retrieved and immunocytochemically stained for the presence of procollagen, a precursor to collagen. Regardless of histocompatibility or preservation, grafts exhibited consistent procollagen presence that equaled or exceeded that seen in the corresponding native valves. Positive procollagen staining was predominantly in the aortic wall. The most prominent staining was near the hinge point of the valve leaflets, with no staining in the free portion of the leaflets. Staining with alpha-actin demonstrated vascular smooth muscle in sites remote from the areas positive for procollagen, which suggests that vascular smooth muscle was not responsible for the procollagen production. These findings indicate that cryopreservation is compatible with persistent fibroblast viability and in vivo protein synthesis by both syngeneic and allogeneic aortic valve grafts.

The importance of thrombus organization and stellate cell phenotype in collagen I gene expression in human, coronary atherosclerotic and restenotic lesions.

OBJECTIVES: Collagen synthesis is one of the major mechanisms of primary atherosclerotic plaque growth and is likely to be similarly important in restenosis. The patterns of collagen gene expression in human restenosis and associations with thrombosis/hemorrhage have not been described. METHODS: Using human coronary artery samples obtained via the atherectomy catheter, we compared primary plaques (40 specimens) and restenotic lesions (41 specimens) for type I collagen gene expression using immunocytochemistry (SPI.D8 antibody to type I procollagen, an intracellular precursor of mature collagen) with subsequent computer image analysis. RESULTS: Scattered positive cells were identified in specific, non-random patterns. According to logistic regression analyses, type I procollagen gene expression seems to be more closely associated with certain morphological features (organized thrombus, microvessels, regions enriched with stellate cells) than with belonging to a primary vs. a restenotic sample. However, there may be a tendency for restenotic tissue to have slightly higher numbers of type I procollagen-positive cells than primary lesion tissue. CONCLUSIONS: Symptomatic primary and restenotic lesions exhibit similar patterns of type I collagen gene expression. Plaque microvessels and thrombi/hemorrhages (common features of both kinds of advanced lesions) might stimulate collagen synthesis equally well irrelevant to the nature of the lesion.

Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Atherosclerosis-prone branch regions in human aorta: microarchitecture and cell composition of intima.

The microarchitecture and cell composition of intima were studied at the macroscopically unaffected branch regions of human thoracic aorta using en face preparations, scanning and transmission electron microscopy, and immunohistochemistry. The endothelial lining showed a heterogeneous pattern and altered morphology including the areas of deendothelialization covered with platelets and dilated intercellular clefts. Leukocyte adhesion, accumulation of subendothelial macrophages and lymphocytes were characteristic of proximal and lateral zones, while the flow divider showed no significant accumulation of blood cells. Smooth muscle cells (SMCs) on the flow divider were elongated, in a contractile state, contacted side-by-side and did not contain lipid inclusions. In the lateral and proximal zones, intima appeared to be a network of stellate SMCs which were in contact through their processes. Most of the SMCs were in a synthetic state and many of them contained small lipid droplets. The number of procollagen I positive cells and the volume of extracellular components were most significant at the lateral zones rather than at the flow divider. We did not observe any difference in the rate of proliferation. Our results suggest that the intimal layer at the lateral and proximal zones has some distinct structural peculiarities, which provoke the development of initial atherosclerotic lesions at these sites. Such an intimal structure is probably caused by different flow patterns at these zone. However, only the totality of different morphological features exhibited in the area of altered vascular wall shear stress may be considered as a prerequisite for atherosclerotic lesions.

Effect of mechanical forces on growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role of individual cell types.

The effect of mechanical stimuli on pulmonary artery growth and matrix tissue synthesis (and how individual cell types in the vessel wall respond to such stimuli) is incompletely characterized. Rabbit pulmonary arteries were placed in tissue culture medium and subjected to varying magnitudes of stretch or hydrostatic pressure (separately) for 4 days. The rate of protein synthesis in smooth muscle cells (by quantitative autoradiography) was positively related to the magnitude of stretch, as were the percentage of procollagen type I-positive cells and the rate of cell replication. In adventitial fibroblasts, stretch increased the rate of replication but not of protein synthesis. Hydrostatic pressure had little or no effect on the variables measured in either smooth muscle cells or fibroblasts. Stretch also increased the rate of elastin and collagen synthesis in the whole pulmonary artery segment, and after 4 days of stretch, the contents of actin and elastin were increased. Removal of the endothelium did not affect stretch-induced protein, collagen, or elastin synthesis but augmented stretch-induced smooth muscle replication. These data suggest that in the intact pulmonary artery, stretch, but not pressure, can stimulate hypertrophy and hyperplasia in smooth muscle cells and hyperplasia in fibroblasts. Matrix protein synthesis and accumulation are also increased by stretch. Neither stretch-mediated growth nor matrix protein synthesis required endothelium in this model.

Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques.

Cell proliferation, an important mechanism of atherosclerotic plaque growth, occurs among smooth muscle, inflammatory cell, and other cell types. We have identified different topographical patterns of cell proliferation in human carotid plaques, based on cell type. Cell proliferation was determined with an antibody to the proliferating cell nuclear antigen (PCNA), combined with cell type-specific antibodies. Despite low levels of overall proliferative activity, the intima displayed more proliferative activity than the underlying media (1.61 +/- 0.35% in intima versus 0.05 +/- 0.03% in media; P < 0.01). The preponderant proliferative cell type in the intima was the monocyte/macrophage (46.0% of PCNA-positive cells), with a minority being smooth muscle alpha-actin-positive (9.7%), microvascular endothelial (14.3%), and T cells (13.1%). Smooth muscle cells were the dominant proliferating cell type in the media (44.4% of PCNA-positive cells versus 20% endothelial cells, 13.0% monocyte/macrophages, and 14.3% T cells). Within the plaque, foam-cell-rich regions mostly displayed proliferation among macrophages (66.5%), whereas in vascularized fields PCNA positivity was almost equally shared by endothelial cells (23.8%), monocyte/macrophages (26.3%), smooth muscle alpha-actin-positive cells (14.0%), and to a lesser extent, T cells (8.2%). Logistic and linear regression analyses also demonstrated that location in foam-cell-rich regions was a significant predictor of proliferation only among monocyte/macrophages, whereas location in vascularized regions was a good predictor of PCNA positivity among both inflammatory and noninflammatory cells. These different patterns of cell type proliferation suggest possibly different distributions of putative responsible growth regulatory factors in human atherosclerosis.

Heterogeneity of smooth muscle cells in embryonic human aorta.

Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for alpha-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.

Three-dimensional organization of biological tissues: a new method for investigation using scanning electron microscopy.

A new method is described which enables the visualization of hidden cellular surfaces of biological tissues. The method does not depend on the kind of fixative solution, the duration of the fixation, or the quantity of extracellular matrix. The samples are chemically dissociated with potassium ethoxide until the first individual cells appear in the solution. This initial process is followed by phase contrast microscopy, and remaining tissue is investigated with scanning electron microscopy. The method has proved more effective than conventional enzymatic techniques and is a simple, quick, and effective way to investigate the three-dimensional organization of different normal and pathological tissues.

Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques?

Previous studies have indicated the focal presence of growth factors and focal low levels of cell proliferation in human atherosclerotic plaques. Using human carotid plaques and an antibody to platelet-derived growth factor (PDGF)-A chain, we have begun to assess growth factor significance by spatially correlating growth factor gene expression with actual cell proliferation. Since PDGF is a mitogen for smooth muscle and related cells and since inflammatory cells (eg, macrophages) can also proliferate in these lesions, it was important to exclude inflammatory cell proliferation from this consideration. Therefore, we have used a triple immunolabeling approach, combining the above anti-PDGF-A chain antibody with an inflammatory cell cocktail (CD68+CD45 for monocyte/macrophages and lymphocytes) and adding an anti-proliferating cell nuclear antigen (PCNA) antibody to mark proliferating cells. In the carotid atherosclerotic plaques, PDGF immunostaining was distributed focally, preferentially in the fibrous cap and vascularized regions, and was present in two distinct patterns: cytoplasmic and diffuse extracellular staining. When we considered colocalization within the same cells, cytoplasmic PDGF-A staining did not appear to colocalize with inflammatory markers. PCNA nuclear staining combined with PDGF cytoplasmic staining of the same cell was detected extremely rarely. Considering colocalization within the same microscopic fields, PDGF-A staining was detected more frequently than noninflammatory PCNA positivity. Quantitative logistic regression analysis demonstrated that localization in vascularized regions and (independently) the presence of PDGF-A are good predictors of noninflammatory cell proliferation, within the same microscopic fields. Therefore, PDGF-A and other factors especially associated with vascularized regions may be involved in the regulation of mesenchymal cell proliferation in human atherosclerotic plaques.

Cell proliferation and collagen synthesis are two independent events in human atherosclerotic plaques.

We have used a double-immunolabelling technique on human carotid atherosclerotic plaques to measure cell proliferation and type-I collagen gene expression, using antibodies to proliferating cell nuclear antigen (PCNA) and type-I procollagen protein, respectively. Although cell proliferative activity and type-I collagen gene expression can occur simultaneously in the same cell, this is a rare event, and the vast majority of collagen-producing cells do not show proliferative activity. These two processes also tend to occur in separate locations, although they can coexist in certain regions of the plaque. This disparate location of these two important modes of plaque growth suggests that cell proliferation and collagen gene expression may be under separate biological controls during the development and evolution of human atherosclerosis.

Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study.

Appearance of contractile filament-laden stromal cells or myofibroblasts is a characteristic of lung fibrotic lesions. The role of these cells in fibrosis and their cytoskeletal phenotype are not fully delineated. This study was undertaken to further investigate these issues using a model of lung fibrosis. Rats were treated endotracheally with bleomycin on day 0, and their lungs examined at various time points by in situ hybridization for alpha 1(I) procollagen mRNA expression and by immunohistochemistry for desmin and alpha-smooth muscle actin expression. The results show an increase in the number of cells resembling fibroblasts and strongly positive for alpha-smooth muscle actin, desmin and procollagen mRNA expression in lungs of animals treated with bleomycin, with the increase being maximal between days 7 and 14 after bleomycin treatment. Two types of newly positive cells could be discerned. The first expressing alpha-smooth muscle actin, desmin, and procollagen mRNA was localized in active fibrotic lesions. The second expressing only alpha-smooth muscle actin and procollagen mRNA was localized in fibrotic submesothelial areas. Almost all of the newly reactive alpha-smooth muscle actin-positive cells strongly express procollagen mRNA, and they constituted most of the cells actively expressing procollagen. These findings suggest that the newly appearing myofibroblast characterized by alpha-smooth muscle actin and/or desmin expression may be responsible for most if not all of the increased lung collagen gene expression in pulmonary fibrosis.